Detection of de-N-glycosylated EPO with SDS-PAGE: A complementary confirmation procedure for recombinant EPO in blood samples.

Frameshift variant c.577del in the EPO gene can result in the extension of the amino acid sequence of EPO by invalidating the termination codon. As the molecular weight of its encoded protein EPO (VAR-EPO) is similar to that of rEPO, the World Anti-Doping Agency has published Annex B to the TD2022EPO in order to protect athletes with variant c.577del from the suspicion of rEPO administrations. However, it is still necessary to develop a confirmation method for rEPO that can discriminate rEPO from VAR-EPO. Based on the glycosylated characteristic of EPO, we selected the detection of de-N-glycosylated EPO as a complementary confirmation method for rEPO in blood samples. All samples were analyzed for both intact EPO and de-N-glycosylated EPO with SDS-PAGE, including rEPO spiked samples and blank samples. The results showed that, after de-N-glycosylation, a single-band was detected in samples collected from non-variant carriers, no matter whether the sample was spiked with rEPO. In samples collected from variant carriers, a double-band was detected. The ratio of lower band to upper band increased significantly corresponding to the concentration of rEPO. We calculated a series of cut-off values by normality distribution function to identify the presence of rEPO. Neither false positive results in blank samples nor false negative results in spiked samples at the applicable Minimum Required Performance Levels were found. This indicates that this method could be adopted as a complementary confirmation method for rEPO in blood samples. A revised testing strategy was also proposed, which would discriminate rEPO directly without further investigation.